Many laboratory tests are determined by how a particular analyte present in sample reacts with a specific reagent. Often, these tests are qualitatively determined by visual inspection. For example, current home pregnancy tests can detect human chorionic gonadotropin (HCG), a hormone secreted in the urine of pregnant women. Typically, the urine is applied to a testing substrate that has an antibody immobilized thereon that is capable of binding to the HCG. A labelled reagent is then applied to the testing substrate that is capable of specifically binding to HCG, and thus, in the presence of HCG, will color the testing substrate, thus indicating a potential pregnancy. For such tests, the user can tell the results at a glance. However, with increasing demand for rapid diagnostic testing in a laboratory setting, visual inspection by a human technician becomes a bottleneck. In addition, human technicians are error prone, especially when performing diagnostic tests that require quantitative measurements of color or optical density. Furthermore, quantifying test results is very difficult for human technicians when the color density of the testing substrate varies due to differences in the amount of reagent used for each test, the amount of liquid (such as blood plasma) deposited on the substrate, or by different batches of reagents used in the test. Additionally, the results of such tests often depend on the difference in color saturation associated with a specific region of the testing substrate where the analyte specifically binds compared to another region of the testing substrate where the analyte is not supposed to bind and thus where the presence of color indicates the degree of "background" or "noise" in the test. This is particularly true for rapid immunoassays that use membranes as the testing substrates that have discrete zones having receptors immobilized thereon that specifically bind to the analyte present in the sample tested.
Consequently, a number of prior art systems have been designed to partially automate the process of quantifying test results. For example, the results of electrophoretic immunoblots (Western Blots), the main method for verification of human immunodeficiency virus seropositivity, can be quantitated using densitometry. Typically with a Western Blot, bands of electrophoresed proteins are transferred to a nitrocellulose strip and then incubated with patient sera. If antibodies specific to the proteins are present in the sera, they will bind to the blotted proteins. The presence of the antibodies can be detected using labelled antibodies to human IgG. If labels are used that generate a visible color, bands will appear that correspond to the location of the blotted proteins for which the patient has antibodies. The concentration of the antibodies can be quantitated using a densitometer, an instrument which measures optical density by measuring the intensity of reflected light. The nitrocellulose strip is passed through a beam of light, so that the intensity of each band is measured and a value is generated that correlates to the concentration of antibody present in the patient sera. However, the densitometer only measures one point of each band as opposed to scanning the entire area of the band. Because the color intensity of a band can vary, as well as the background color surrounding each band, the values generated by densitometry can vary and thus may not accurately reflect the true concentration of the substance being measured.
Reflectormeters are also used to quantitate the results of certain laboratory tests, particularly, rapid immunoassays such as those described in U.S. Pat. Nos. 5,006,464 to Chu etal. and 4,632,901 to Valkirs et al., which, after the performance of assay steps, can result in the appearance of a colored region on a testing substrate to indicate the presence of a particular analyte in a sample. The reflectometer is a photoelectric instrument for measuring the optical reflectance of a surface. Typically, the rapid immunoassay comprises a testing substrate such as a porous membrane. A small portion of the testing substrate has a receptor immobilized thereon (i.e. the receptor area--usually a small circular area or dot) that is capable of binding directly or indirectly to an analyte such as an antibody, protein, hormone, or any other substance that is suspected of being present in a patient sample. Thus, when a patient sample, such as plasma or urine, comes in contact with the testing substrate, the analyte, if present in the sample, will bind specifically to the receptor area of the testing substrate, but not to the peripheral area of the testing substrate where no receptor is immobilized. The remainder of the sample and any unbound analyte will flow through the testing substrate, if it is porous, and/or can be washed off. A labeled reagent is added that is capable of binding directly or indirectly to the analyte to generate a colored dot or circle (or whatever shape the receptor area is). Thus, if the analyte is present in the patient sample, it will bind to the receptor area and its presence will be indicated by the generation of color after application of the labeled reagent.
The results of the immunoassay can then be measured using a reflectometer. Typically, the testing substrate needs to be inserted into the reflectometer so that the receptor area will align with a beam of light that is used to measure reflectance. Therefore, if the receptor area is not accurately positioned on the testing substrate, the results of the assay will not be accurately measured. Additionally, there may be variation in the color intensity generated at the receptor area. Thus, the beam of light may not line up with the part of the receptor area that most accurately correlates to the concentration of analyte present in the patient sample.
Digital analysis has been used in some testing procedures, but has not been used for quantifying the results of immunoassays. For example, U.S. Pat. No. 5,018,209 issued May 21, 1991 and U.S. Pat. No. 5,008,185 issued Apr. 16, 1991, both to Bacus, describe digital image processing methods and apparatus to analyze various features of cells being viewed on a slide under a microscope. Because the cells (or portions thereof) are randomly located on the slide, the technician and system work in an interactive fashion whereby the technician manually locates the cells on the slide that the system thereafter analyzes. Thus, while the efficiency of the testing process is increased by such an interactive system, it is not as efficient as one which would automatically locate the region of interest without human interaction.
U.S. Pat. No. 4,922,915 issued May 8, 1990 to Arnold, describes an automatic image location method in the field of medical imaging technology, such as computer tomography (CT) and magnetic resonance imaging (MRI). In a typical diagnostic scan of a patient, several reference samples of known optical density are placed in proximity with the patient's body and are scanned simultaneously. These images of the reference samples of known density are compared with the images of various regions of the patient's body to determine the relevant characteristics of those regions.
The method in Arnold is concerned with locating two regions: the reference samples and the regions of interest within the patient's body. With respect to the reference samples, the system locates the samples automatically by two separate algorithms. The first algorithm uses the fact that the reference samples are of known optical densities. The Arnold system searches the entire digital image for regions with these optical densities.
The second location algorithm uses pre-positioned metallic rods proximately placed to the reference samples. Initially, the system starts scanning the entire digital image for pixels of greatest density. These pixels correspond to the metallic rods. Once the rods are located, the reference samples are easily located because the orientation of the samples in relation to the metallic rods is predefined.
With respect to locating regions of interest in the patient's body, the search performed by the Arnold system is not fully automated. After the reference samples are located, Arnold requires that a human operator define an enlarged region of interest, for example around a bone structure, which the system thereafter refines. This step in Arnold is necessary because the system is unable to exclude regions which add error to the density readings.
While Arnold's method of automatically locating digital images works well when regions are either of known densities or known orientations, it is not satisfactory when the region of interest has neither known intensity or position. In Arnold's method, human interaction during the analysis step is always required.
The above-mentioned methods of digital analysis do not preform quantitative analyses of specimens, but rather only locate a region of interest based on optical density. However, in the analysis of chemical and biological specimens, it is often the density of a particular color that is the relevant measurement parameter. For instance, some immunoassays employ labeled reagents, such as certain colloidal reagents, that generate a color when an analyte is detected in a biological specimen.
Therefore, it is an object of the present invention to provide a system and method for automatic image location and quantitative analysis when the region of interest is of neither known intensity or position in the image.
Another object of the present invention is to provide a reliable automated method for quantifying the results of an immunoassay wherein the method provides a more accurate measurement that better corresponds to the true concentration of an analyte in a fluid sample compared with prior art methods that use densitometers and reflectometers.